Repositório Colecção:http://hdl.handle.net/10362/10532018-02-22T06:49:28Z2018-02-22T06:49:28ZRegeneration Studies in the African Spiny MousePrata, Diogo Filipe Cabritahttp://hdl.handle.net/10362/308882018-02-22T03:05:41Z2017-11-01T00:00:00ZTítulo: Regeneration Studies in the African Spiny Mouse
Autor: Prata, Diogo Filipe Cabrita
Resumo: Across the Metazoa, organisms vary in terms of how they respond to injury. The two basic responses are regeneration or fibrotic scarring. While some groups such as axolotls, amphibians and fish show high regenerative capacity, mammals tend to heal wounds by fibrotic scarring. The African Spiny Mouse (Acomys) has been reported to have the capacity of closing 4-mm full thickness wounds in the ear pinna with full regeneration of the original tissue architecture, including dermis, epidermis, cartilage and hair follicles. In contrast, Mus musculus heals the border wounds by fibrotic scarring. Therefore, Acomys and Mus constitute a powerful comparative framework for the study of mammalian regeneration.
The goal in this work was to answer two independent questions.
First, we asked whether mesenchymal stem cells (MSC) were present in ear tissue of both species and compared their differentiation capabilities in vitro. Primary cell cultures were established from uninjured ears of both species, immune-phenotyped and cultured in vitro in adipocyte, chondrocyte and osteocyte differentiation media. Differentiation was characterized by staining and marker expression. We found that Mus cells tend to differentiate to adipocytes, while Acomys cells tend to differentiate to chondrocytes.
Second, we asked whether telomerase was differentially upregulated in Acomys vs Mus in response to wounding. Both species were subjected to ear wounds and allowed to heal or regenerate. Tissues were harvested at different time points and analyzed for TERT expression by RT-qPCR. Our results were inconclusive.
This work constitutes a further step in understanding the molecular and cellular mechanisms that distinguish Acomys as an emerging mammalian regeneration model.2017-11-01T00:00:00ZA importância da fase pré-analítica em determinações genéticasFrade, Ana Rita Teixeirahttp://hdl.handle.net/10362/308872018-02-20T10:29:09Z2017-11-01T00:00:00ZTítulo: A importância da fase pré-analítica em determinações genéticas
Autor: Frade, Ana Rita Teixeira
Resumo: Este Relatório de Atividade Profissional tem por objetivo a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa.
O documento apresentado centra-se na importância da fase pré-analítica, contemplando todas as suas etapas: colheita de sangue, utilizando o anticoagulante adequado para futura utilização das amostras em técnicas de amplificação de DNA, cuidados a ter com o armazenamento, acondicionamento e transporte das amostras. A relevância deste tema decorre do facto de a candidata exercer a profissão de Técnica de Análises Clínicas e Saúde Pública num laboratório de Patologia Clínica hospitalar que não realiza determinações analíticas por biologia molecular, sendo as colheitas efetuadas enviadas para laboratórios situados em regiões geográficas muito afastadas.
Assim, neste trabalho é exposta e analisada a forma como o laboratório onde exerce a profissão executa a fase pré-analítica, baseando-se nas regras de boas práticas do laboratório e noutras especificações fornecidas pelos laboratórios recetores. São enquadradas as falhas que podem ocorrer em cada uma das etapas referidas e discutidas as soluções implementadas para a sua resolução. Noutro âmbito, são apresentados exemplos concretos de analitos e a sua utilização posterior em ensaios de biologia molecular, evidenciando as interdependências entre as duas fases, ou seja, a forma como a fase pré- analítica condiciona a aplicação das metodologias subsequentes e os seus resultados. Neste contexto, é fornecido em detalhe o protocolo utilizado por um dos laboratórios recetores na determinação genética da resistência aos antirretrovirais pelo vírus da imunodeficiência humana tipo I.
A finalizar, é discutida a forma como os erros associados à fase pré-analítica têm sido minimizados com a implementação de sistemas informáticos nos laboratórios e poderão vir a sê-lo mais ainda com a implementação nos mesmos de sistemas de garantia da qualidade como a Acreditação e a Certificação e ações de formação interna dirigidas a todos os profissionais de saúde que intervêm no processo.2017-11-01T00:00:00ZTowards determining the structure of human KIFC1, a potential target for drug development in cancer therapyViegas, Mariana Filipa de Almeidahttp://hdl.handle.net/10362/279082018-01-15T06:23:48Z2017-11-01T00:00:00ZTítulo: Towards determining the structure of human KIFC1, a potential target for drug development in cancer therapy
Autor: Viegas, Mariana Filipa de Almeida
Resumo: Normal cell division is initiated upon centrosome duplication and the two centrosomes move towards the cell periphery to form the poles of a bipolar mitotic spindle, a function that is essential for accurate chromosome segregation. Aberrant centrosomes duplication leading to more than two centrosomes is referred to as centrosome amplification. Centrosome amplification causes genetic instability and is a hallmark in cancer cells. Cancer cells survive the multiple mitotic spindle formation by clustering Microtubules Organizing Centres (MTOCs) to form two supernumerary centrosomes and a single pseudo-bipolar spindle during metaphase. KIFC1, a kinesin essential for this process, is particularly interesting as a potential therapeutic target. This is due to its non-essential role in cell division in normal cells but crucial centrosome clustering function in cancer cells with supernumerary centrosomes.
The overall goal of this project was to determine the structure of the catalytic domain, also known as motor domain, of KIFC1 to aid future structure-based drug-design.
In this project, I have established the expression and purification protocol for the KIFC1 motor domain. The protocol enables to obtain large amounts of protein at highest purity. Although I obtained crystals initially, their reproduction was difficult and therefore high-resolution crystals of the KIFC1 motor domain proved difficult to obtain. As such, the search for crystallisation conditions that allow the formation of high-resolution and reproducible crystals of the KIFC1 motor domain continues. Once this bottleneck is surpassed, it will be possible to use the structure for future structure-based drug-design.2017-11-01T00:00:00ZCaffeine Effects on the Gene Expression Profile of iPSC-derived Neuronal CellsDias, Marta Isabel Silvahttp://hdl.handle.net/10362/278462018-01-10T04:07:38Z2017-12-01T00:00:00ZTítulo: Caffeine Effects on the Gene Expression Profile of iPSC-derived Neuronal Cells
Autor: Dias, Marta Isabel Silva
Resumo: Caffeine is widely and massively consumed on daily basis in the form of coffee, tea or energy drinks. This very popular, psychoactive drug is sought after due to its ability to increase energy and alertness, enhance physical and cognitive performance as well as to improve our ability of focusing. Previous studies have investigated the ability of caffeine to inhibit the activation of adenosine receptors in low doses, amongst other pharmacological effects as well as its potential in several neurodegenerative disease-modeling studies. However, not much is known about the effects that caffeine exerts on gene expression of neuronal cells.
In this study we aim to understand if and how physiological concentrations of caffeine affect gene expression in human induced pluripotent stem cell (iPSC)-derived neuronal cells. Moreover, we identify active enhancers in neuronal cells and investigate the extent to which enhancers might be involved in the regulation of neuronal responses to caffeine.
Using Cap Analysis of Gene Expression (CAGE) RNA expression profiling, we obtained a comprehensive data set of transcription start sites and enhancer activity of neuronal cells exposed to various caffeine concentrations (0, 3 and 10 μM).
We identified a set of genes that appears to be involved in the mediation of caffeine response. Synaptic activity is upregulated after 1 hour of 3 μM caffeine treatment as well as dopaminergic neurotransmission. Immune system processes as well as axon guidance events are downregulated after 3 hours of 10 μM caffeine exposure.
These insights provide concrete hypotheses of physiological processes and associated genes for guiding further functional validation experiments with the potential to give valuable insights into the effects of caffeine to the human brain.2017-12-01T00:00:00Z